The goal of this proposal is to identify novel, activity-dependent mechanisms by which microglia interact with neural circuits and regulate plasticity of synapses. Neuronal activity is a critical regulator of synaptic architecture. Both in the developing brain and during adulthood, changes in activity drives the formation of new synapses, elimination of less active synapses, and maintenance of more active synapses. Despite over 50 years of research, the mechanisms by which neuronal activity drives changes in synaptic connectivity remain to be fully deciphered. Unexpectedly, we discovered that microglia, resident central nervous system (CNS) macrophages, are regulated by neuronal activity and engulf and prune away less active synapses in the healthy developing visual system. Further studies have identified similar microglial synaptic pruning functions in other brain regions as well as functions in synaptogenesis and neural transmission. These data compel us to consider microglia as key cellular regulators of synaptic connectivity and evoke the paradigm shifting possibility that disruptions in microglial function result in synaptic defects (density, transmission, etc.) in a wide range of neurological disorders ranging from autism to Alzheimer?s disease. Here, we propose that changes in neuronal activity elicit activity-dependent genetic programs in microglia necessary for regulating synaptic connectivity. We will focus our analyses on microglia-mediated synaptic pruning. In support of this hypothesis, we recently silenced excitatory neurons in the adult brain using designer receptors exclusively activated by designer drugs (DREADDS) followed by microglia-specific translating ribosomal affinity purification and RNA sequencing (TRAP-seq). Sequencing results revealed rapid induction of phagocytic pathways in microglia following acute neuronal silencing. We will now use our newly developed in vitro assays to molecularly dissect the function of these phagocytosis-related genes in microglia-dependent synaptic engulfment (Aim 1). To identify new molecules relevant to activity-dependent synaptic pruning during brain development, we will also perform microglia-specific TRAP-seq in a paradigm we recently identified to robustly upregulate microglial synaptic engulfment in response to changes in sensory experience in vivo?whisker deprivation-induced synapse elimination in the mouse barrel cortex (Aim 2). We will again use our in vitro assays to assess microglial synaptic engulfment and validate gene hits. Using our combined expertise in cell- specific TRAP-seq (A. Schaefer lab) and microglial synaptic pruning (D. Schafer lab), we have a unique and powerful advantage to identify novel mechanisms regulating activity-dependent microglia-synapse interactions necessary for modulating synaptic connectivity. Long term, we will collaborate to explore these mechanisms in vivo and determine whether mechanisms are dysregulated in neuropsychiatric disorders with known defects in microglial inflammatory state and synaptic connectivity.